Project Summary/Abstract Hypertrophic cardiomyopathy (HCM) is a heritable cardiovascular disease that is the leading cause of sudden cardiac death in young adults. More than half of all HCM patients are identified to carry missense mutations in genes encoding saromeric proteins, predominantly human ?-cardiac myosin, the thick filament motor that powers ventricular contraction. Current treatment for HCM is limited to symptomatic relief. It is pressing to understand how HCM-causing mutations in human ?-cardiac myosin alter the biomechanical function of the motor protein at the molecular level, which is a necessary prerequisite for the development of targeted therapies. Recent biochemical and biophysical studies using recombinant human ?-cardiac myosin suggest that early-onset HCM-causing mutations significantly increase the power output of the motor by increasing velocity, intrinsic force, and ATPase activity, consistent with clinical observations that HCM-causing mutations lead to hyper-contractility of the heart muscle. However, similar studies of mutations that give rise to severe disease in adulthood have shown only subtle effects on these parameters. An overlooked parameter in the biomechanical function of the myosin motor protein is the number of myosin heads functionally available for interaction with actin (Na). CryoEM studies of striated muscle myosins suggest that myosin heads (S1) may fold back and interact with their proximal tail region (proxS2) and with each other. The only functional data to support this idea are from the Spudich lab demonstrating that recombinant human ?-cardiac myosin S1 can bind to proxS2 in a salt-dependent manner. We hypothesize that this intra-molecular interaction possibly sequester myosin heads and prevent them from interacting with actin, thus regulating Na and imparting fine-tuned control of cardiac contractility. HCM-causing mutations located on the interacting surfaces will weaken this interaction and lead to an increase in Na, thus freeing myosin heads to interact with actin and causing hyper-contractility. To test this hypothesis, I propose to (1) Measure changes in the binding affinities between S1 myosin head and proxS2 myosin tail induced by HCM-causing mutations using Microscale Thermophoresis, (2) Directly visualize conformational change of human ?-cardiac myosin between an open and a sequestered state using a novel approach based on single-molecule fluorescence resonance energy transfer, and (3) Determine the effects of HCM-causing mutations on sequestration-dependent changes in the actin-activated ATPase activity of human ?-cardiac myosin. Our results will provide a more comprehensive understanding on how HCM-causing mutations affect the function of human ?-cardiac myosin to generate power by determining whether these mutations alter the ability of myosin to adopt a sequestered conformation. Ultimately, this research will have a significant impact on the development of small molecule drugs targeted on specific changes in the structure and functions of the cardiac myosin induced by the disease-causing mutations.